Received May 18, 2018
Recombinant human erythropoietin (EPO) with additional N-terminal
heparin-binding protein domain (HBD) from bone morphogenetic protein 2
was synthesized in Escherichia coli cells. A procedure for
HBD-EPO purification and refolding was developed for obtaining
highly-purified HBD-EPO. The structure of recombinant HBD-EPO was close
to that of the native EPO protein. HBD-EPO contained two disulfide
bonds, as shown by MALDI-TOF mass spectrometry. The protein
demonstrated in vitro biological activity in the proliferation
of human erythroleukemia TF-1 cell test and in vivo activity in
animal models. HBD-EPO increased the number of reticulocytes in the
blood after subcutaneous injection and displayed local angiogenic
activity after subcutaneous implantation of demineralized bone matrix
(DBM) discs with immobilized HBD-EPO. We developed a quantitative
sandwich ELISA method for measuring HBD-EPO concentration in solution
using rabbit polyclonal serum and commercial monoclonal anti-EPO
antibodies. Pharmacokinetic properties of HBD-EPO were typical for
bacterially produced EPO. Under physiological conditions, HBD-EPO can
reversibly bind to DBM, which is often used as an osteoplastic material
for treatment of bone pathologies. The data on HBD-EPO binding to DBM
and local angiogenic activity of this protein give hope for successful
application of HBD-EPO immobilized on DBM in experiments on bone
regeneration.
KEY WORDS: erythropoietin, Escherichia coli,
heparin-binding domain